Chronic visceral pain is a significant problem in the US and current treatments are largely ineffective. Why visceral pain is so poorly managed is unknown, but we have demonstrated that adult male and female rats develop visceral hypersensitivity to colonic distension after chronic psychological stress, with a significantly exaggerated response in females. Moreover, we found that female rats exposed to unpredictable early life stress (ELS) exhibit visceral hypersensitivity as adults, whereas males have no evidence of increased colonic sensitivity. Together, this evidence points to a sexually dimorphic female vulnerability to stress-induced colonic hypersensitivity. Our preliminary data also indicates that epigenetic modifications contribute to stress-induced visceral pain through alterations in glucocorticoid receptor (GR) and corticotropin-releasing hormone (CRH) expression in the central nucleus of the amygdala (CeA). However, three important questions still remain: 1) Do sexually dimorphic differences in the expression of GR and CRH within the CeA explain why females are more susceptible to chronic stress-induced visceral pain? 2) Do specific epigenetic mechanisms in the CeA explain why exposure to unpredictable ELS confers visceral pain vulnerability in female rats while male rats are resilient? 3) Is the CeA the fulcrum for the development of stress-induced visceral pain. To provide answers Aim 1 will utilize state-of-the-art approaches to focus on epigenetic dysregulation in the CeA that mediate the persistent sexually dimorphic effects of adult stress on hypersensitivity. Aim 2 we will take advantage of pivotal preliminary data to test the hypothesis that there are sexually dimorphic epigenetic mechanisms within the CeA conferring visceral pain vulnerability in adult females following unpredictable ELS. Aim 3 will determine whether stress-induced changes in visceral nociceptive processing are specific to the amygdala. Taken together we are proposing a novel approach by using state-of-the-art epigenetic techniques combined with behavioral out- comes to identify mechanisms to enhance our basic understanding of chronic visceral pain. Specific Aim 1 will show that heightened visceral pain following chronic adult stress is modulated through epigenetic mechanisms within the CeA. Specific Aim 2 will show that as adults, female rats exposed in unpredictable ELS have an epigenetic signature in the CeA that differs from male rats and aim 3 will investigate the amygdala specificity of our findings. This application will build upon our novel preliminary findings and take full advantage of cutting- edge approaches to delineate central molecular mechanisms leading to stress-induced chronic visceral pain. RELEVANCE TO HUMAN HEALTH: This NIH R01 grant will substantially advance our knowledge of the neural and molecular level events responsible for chronic pain, and with our previous data provide a unifying hypothesis for how stress leads to chronic pain. Scientifically, the results will identify novel sexually dimorphic mechanisms and circuitry involved in female vulnerability to develop chronic stress-induced pain, leading to new targets for therapies to treat chronic pain, which is a significant unmet healthcare burden.